The present invention relates to a method of producing phytase in yeast, yeast strains which express heterologous phytase, and the heterologous phytase produced by yeast.
Phytases, a specific group of monoester phosphatases, are required to initiate the release of phosphate (xe2x80x9cPxe2x80x9d) from phytate (myo-inositol hexophosphate), the major storage form of P in cereal foods or feeds (Reddy, N. R. et al., xe2x80x9cPhytates in Legumes and Cereals,xe2x80x9d Advances in Food Research, 28:1 (1982)). Because simple-stomached animals like swine and poultry as well as humans have little phytase activity in their gastrointestinal tracts, nearly all of the ingested phytate P is indigestible. This results in the need for supplementation of inorganic P, an expensive and non-renewable nutrient, in diets for these animals. More undesirably, the unutilized phytate-P excreted through manure of these animals becomes P pollution of the environment (Cromwell, G. L. et al., xe2x80x9cPxe2x80x94A Key Essential Nutrient, Yet a Possible Major Pollutantxe2x80x94Its Central Role in Animal Nutrition,xe2x80x9d Biotechnology In the Feed Industry; Proceedings Alltech 7th Annual Symposium, p. 133 (1991)). Furthermore, phytate chelates with essential trace elements like zinc and produces nutrient deficiencies such as growth and mental retardation in children ingesting mainly plant origin foods without removal of phytate.
Two phytases, phyA and phyB, from Aspergillus niger NRRL3 135 have been cloned and sequenced (Ehrlich, K. C. et al., xe2x80x9cIdentification and Cloning of a Second Phytase Gene (phys) from Aspergillus niger (ficuum),xe2x80x9d Biochem. Biophys. Res. Commun., 195:53-57 (1993); Piddington, C. S. et al., xe2x80x9cThe Cloning and Sequencing of the Genes Encoding Phytase (phy) and pH 2.5-optimum Acid Phosphatase (aph) from Aspergillus niger var. awamori,xe2x80x9d Gene, 133:56-62 (1993)). Recently, new phytase genes have been isolated from Aspergillus terreus and Myceliophthora thermophila (Mitchell et al., xe2x80x9cThe Phytase Subfamily of Histidine Acid Phosphatases: Isolation of Genes for Two Novel Phytases From the Fungi Aspergillus terreus and Myceliophthora thermophila,xe2x80x9d Microbiology 143:245-252, (1997)), Aspergillus fumigatus (Pasamontes et al., xe2x80x9cGene Cloning, Purification, and Characterization of a Heat-Stable Phytase from the Fungus Aspergillus fumigatusxe2x80x9d Appl. Environ. Microbiol., 63:1696-1700 (1997)), Emericella nidulans and Talaromyces thermophilus (Pasamontes et al., xe2x80x9cCloning of the Phytase from Emericella nidulans and the Thermophilic Fungus Talaromyces thermophilus,xe2x80x9d Biochim. Biophys. Acta., 1353:217-223 (1997)), and maize (Maugenest et al., xe2x80x9cCloning and Characterization of a cDNA Encoding a Maize Seedling Phytase,xe2x80x9d Biochem. J. 322:511-517, 1997)).
Various types of phytase enzymes have been isolated and/or purified from Enterobacter sp.4 (Yoon et al., xe2x80x9cIsolation and Identification of Phytase-Producing Bacterium, Enterobacter sp. 4, and Enzymatic Properties of Phytase Enzyme.,xe2x80x9d Enzyme and Microbial Technology 18:449-454 (1996)), Klebsiella terrigena (Greiner et al., xe2x80x9cPurification and Characterization of a Phytase from Klebsiella terrigena.,xe2x80x9d Arch. Biochem. Biophys. 341:201-206 (1997)), and Bacillus sp. DS11 (Kim et al., xe2x80x9cPurification and Properties of a Thermostable Phytase from Bacillus sp. DS11,xe2x80x9d Enzyme and Microbial Technology 22:2-7 (1998)). Properties of these enzyme have been studied. In addition, the crystal structure of phy A from Aspergillus ficuum has been reported (Kostrewa et al., xe2x80x9cCrystal Structure of Phytase from Aspergillus ficuum at 2.5 A Resolution,xe2x80x9d Nature Structure Biology 4:185-190 (1997)).
Hartingsveldt et al. introduced phyA gene into A. niger and obtained a ten-fold increase of phytase activity compared to the wild type. (xe2x80x9cCloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus Niger,xe2x80x9d Gene 127:87-94 (1993)). Supplemental microbial phytase of this source in the diets for pigs and poultry has been shown to be effective in improving utilization of phytate-P and zinc (Simons et al., xe2x80x9cImprovement of Phosphorus Availability By Microbial Phytase in Broilers and Pigs,xe2x80x9d Br. J. Nutr., 64:525 (1990); Lei, X. G. et al., xe2x80x9cSupplementing Corn-Soybean Meal Diets With Microbial Phytase Linearly Improves Phytate P Utilization by Weaning Pigs,xe2x80x9d J. Anim. Sci., 71:3359 (1993); Lei, X. G. et al., xe2x80x9cSupplementing Corn-Soybean Meal Diets With Microbial Phytase Maximizes Phytate P Utilization by Weaning Pigs,xe2x80x9d J. Anim. Sci., 71:3368 (1993); Cromwell, G. L. et al., xe2x80x9cPxe2x80x94A Key Essential Nutrient, Yet a Possible Major Pollutantxe2x80x94Its Central Role in Animal Nutrition,xe2x80x9d Biotechnology In the Feed Industry; Proceedings Alltech 7th Annual Symposium, p. 133 (1991)). But, expenses of the limited available commercial phytase supply and the activity instability of the enzyme to heat of feed pelleting preclude its practical use in animal industry (Jongbloed, A. W. et al., xe2x80x9cEffect of Pelleting Mixed Feeds on Phytase Activity and Apparent Absorbability of Phosphorus and Calcium in Pigs,xe2x80x9d Animal Feed Science and Technology, 28:233-242 (1990)). Moreover, phytase produced from A. niger is presumably not the safest source for human food manufacturing.
Yeast can be used to produce enzymes effectively while grown on simple and inexpensive media. With a proper signal sequence, the enzyme can be secreted into the media for convenient collection. Some yeast expression systems have the added advantage of being well accepted in the food industry and are safe and effective producers of food products.
Pichia pastoris is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. This system is well-known for its ability to express high levels of heterologous proteins. Because it is an eukaryote, Pichia has many of the advantages of higher eukaryotic expression systems such as protein processing, folding, and post-transcriptional modification.
Thus, there is a need to develop an efficient and simple system to produce phytase economically for the application of food and feed industry.
The present invention relates to a method of producing phytase in yeast by introducing a heterologous gene which encodes a protein or polypeptide with phytase/acid phosphatase activity into a yeast strain and expressing that gene.
The present invention also relates to a protein or polypeptide having phytase activity with optimum activity in a temperature range of 57-65xc2x0 C. at pH of 2.5 to 3.5 or of 5.5. Optimal pH at 2.5 to 3.5 is particularly important for phytase, because that is the stomach pH of animals.
The invention further provides a yeast cell carrying a heterologous gene which encodes a protein or polypeptide with phytase activity and which is functionally linked to a promoter capable of expressing phytase in yeast.
Yet another aspect of the invention is a vector having a gene from a non-yeast organism which encodes a protein or polypeptide with phytase activity, a promoter which is capable of initiating transcription in yeast functionally linked to the gene encoding a peptide with phytase activity, and with an origin of replication capable of maintaining the vector in yeast or being capable of integrating into the host genome.
The invention also provides a method for producing a protein or polypeptide having phytase activity. An isolated appA gene, which encodes a protein or polypeptide with phytase activity, is expressed in a host cell.
The invention also includes a method of converting phytate to inositol and inorganic phosphate. The appA gene expresses a protein of polypeptide with phytase activity in a host cell. The protein or polypeptide is then contacted with phytate to catalyze the conversion of phytate to inositol and inorganic phosphate.